METHODS FOR THE GENETIC ENGINEERING OF MAMMALIAN CELLS USING SERIAL SUPPLY

Claims

This description relates to methods for improving the efficiency of gene editing in mammalian cells by serially delivering the gene-editing machinery and an incubation period between delivery stages that can range from approximately 1 hour to approximately 60 hours. The broad compatibility of these improved methods is demonstrated with multiple independent lines of induced pluripotent stem cells (iPSCs).

1. A method for generating induced pluripotent stem cells (iPSCs) by genetic engineering comprising: a) supplying a first payload comprising DNA to an iPSC culture; b) incubating the iPSC culture in a medium containing the first payload for approximately 12-60 hours; c) supplying the iPSC culture with a second payload comprising: an RNA and a nuclease, or an RNA and a nucleotide sequence encoding a nuclease; thereby mediating single- or double-strand DNA breakage at a target locus; and d) further incubating the iPSC culture for approximately 12-60 hours, thereby producing iPSCs by genetic engineering.

23. A method for generating human induced pluripotent stem cells (hiPSCs) by genetic engineering, comprising: a) delivering a first load to a hiPSC culture, wherein the first load comprises a donor nucleotide sequence comprising a knock-in nucleotide sequence, and wherein the first load is delivered using nucleofection; b) incubating the hiPSC culture with the first load for approximately 18-30 hours; and c) delivering a second load to the hiPSC culture, wherein the second load comprises Cas12a (Cpf1), Cas9, or Cas13, or a homolog or functional variant thereof, and a guide sequence that is complementary to a target nucleotide sequence; thereby producing hiPSCs by genetic engineering.

28. A method for generating mammalian cells by genetic engineering comprising: a) supplying a first load comprising DNA to a mammalian cell culture; b) incubating the mammalian cell culture in a medium containing the first load for approximately 12-60 hours; and c) supplying the mammalian cell culture with a second load comprising an RNA, a nuclease, and / or a nucleotide sequence encoding a nuclease, thereby mediating single- or double-strand DNA breakage at a target locus; and d) further incubating the mammalian cell culture for approximately 12-60 hours, thereby producing mammalian cells by genetic engineering.